Methods for analyzing nucleic acid sequences

ABSTRACT

The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

CROSS-REFERENCE

This application is a continuation application of Ser. No. 09/572,530,filed May 17, 2000, now U.S Pat. No. 7,056,661 which in turn claimspriority to provisional application Ser. No. 60/134,827 filed May 19,1999. These priority applications are incorporated herein by referencein their entirety.

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 60/134,827, filed May 19, 1999.

This invention was made with funds provided by the U.S. Government underNational Science Foundation Grant No. BIR8800278, National Institutes ofHealth Grant No. P412RR04224-11, and Department of Energy Grant No.066898-0003891. The U.S. Government may have certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to a method for determining the sequenceof nucleic acid molecules.

BACKGROUND OF THE INVENTION

The goal to elucidate the entire human genome has created an interest intechnologies for rapid DNA sequencing, both for small and large scaleapplications. Important parameters are sequencing speed, length ofsequence that can be read during a single sequencing run, and amount ofnucleic acid template required. These research challenges suggest aimingto sequence the genetic information of single cells without prioramplification, and without the prior need to clone the genetic materialinto sequencing vectors. Large scale genome projects are currently tooexpensive to realistically be carried out for a large number oforganisms or patients. Furthermore, as knowledge of the genetic basisfor human diseases increases, there will be an ever-increasing need foraccurate, high-throughput DNA sequencing that is affordable for clinicalapplications. Practical methods for determining the base pair sequencesof single molecules of nucleic acids, preferably with high speed andlong read lengths, would provide the necessary measurement capability.

Two traditional techniques for sequencing DNA are the dideoxytermination method of Sanger (Sanger et al., Proc. Natl. Acad. Sci.U.S.A. 74: 563-5467 (1977)) and the Maxam-Gilbert chemical degradationmethod (Maxam and Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74: 560-564(1977)). Both methods deliver four samples with each sample containing afamily of DNA strands in which all strands terminate in the samenucleotide. Ultrathin slab gel electrophoresis, or more recentlycapillary array electrophoresis is used to resolve the different lengthstrands and to determine the nucleotide sequence, either bydifferentially tagging the strands of each sample before electrophoresisto indicate the terminal nucleotide, or by running the samples indifferent lanes of the gel or in different capillaries. Both the Sangerand the Maxam-Gilbert methods are labor- and time-intensive, and requireextensive pretreatment of the DNA source. Attempts have been made to usemass spectroscopy to replace the time-intensive electrophoresis step.For review of existing sequencing technologies, see Cheng “High-SpeedDNA-Sequence Analysis,” Prog. Biochem. Biophys. 22: 223-227 (1995).

Related methods using dyes or fluorescent labels associated with theterminal nucleotide have been developed, where sequence determination isalso made by gel electrophoresis and automated fluorescent detectors.For example, the Sanger-extension method has recently been modified foruse in an automated micro-sequencing system which requires onlysub-microliter volumes of reagents and dye-labelleddideoxyribonucleotide triphosphates. In U.S. Pat. No. 5,846,727 to Soperet al., fluorescence detection is performed on-chip with one single-modeoptical fiber carrying the excitation light to the capillary channel,and a second single-mode optical fiber collecting the fluorescentphotons. Sequence reads are estimated in the range of 400-500 baseswhich is not a significant improvement over the amount of sequenceinformation obtained with traditional Sanger or Maxam-Gilbert methods.Furthermore, the Soper method requires PCR amplification of templateDNA, and purification and gel electrophoresis of the oligonucleotidesequencing ‘ladders,’ prior to initiation of the separation reaction.These systems all require significant quantities of target DNA. Even themethod described in U.S. Pat. No. 5,302,509 to Cheeseman, which does notuse gel electrophoresis for sequence determination, requires at least amillion DNA molecules.

In a recent improvement of a sequencing-by-synthesis methodologyoriginally devised ten years ago, DNA sequences are being deduced bymeasuring pyrophosphate release upon testing DNA/polymerase complexeswith each deoxyribonucleotide triphosphate (dNTP) separately andsequentially. See Ronaghi et al., “A Sequencing Method Based onReal-Time Pyrophosphate,” Science 281: 363-365 (1998) and Hyman, “A NewMethod of Sequencing DNA,” Anal. Biochem. 174: 423-436 (1988). Whileusing native nucleotides, the method requires synchronization ofpolymerases on the DNA strands which greatly restricts sequence readlengths. Only about 40 nucleotide reads were achieved, and it is notexpected that the detection method can approach single moleculesensitivity due to limited quantum efficiency of light production byluciferase in the procedure presented by Ronaghi et al., “A SequencingMethod Based on Real-Time Pyrophosphate,” Science 281: 363-365 (1998).Furthermore, the overall sequencing speed is limited by the necessarywashing steps, subsequent chemical steps in order to identifypyrophosphate presence, and by the inherent time required to test eachbase pair to be sequenced with all the four bases sequentially. Also,difficulties in accurately determining homonucleotide stretches in thesequences were recognized.

Previous attempts for single molecule sequencing (generally unsuccessfulbut seminal) have utilized exonucleases to sequentially releaseindividual fluorescently labelled bases as a second step after DNApolymerase has formed a complete complementary strand. See Goodwin etal., “Application of Single Molecule Detection to DNA Sequencing,”Nucleos. Nucleot. 16: 543-550 (1997). It consists of synthesizing a DNAstrand labelled with four different fluorescent dNTP analogs, subsequentdegradation of the labelled strand by the action of an exonuclease, anddetection of the individual released bases in a hydrodynamic flowdetector. However, both polymerase and exonuclease have to show activityon a highly modified DNA strand, and the generation of a DNA strandsubstituted with four different fluorescent dNTP analogs has not yetbeen achieved. See Dapprich et al., “DNA Attachment to Optically TrappedBeads in Microstructures Monitored by Bead Displacement,” Bioimaging 6:25-32 (1998). Furthermore, little precise information is known about therelation between the degree of labeling of DNA and inhibition ofexonuclease activity. See Dörre et al., “Techniques for Single MoleculeSequencing,” Bioimaging 5: 139-152 (1997).

In a second approach utilizing exonucleases, native DNA is digestedwhile it is being pulled through a thin liquid film in order tospatially separate the cleaved nucleotides. See Dapprich et al., “DNAAttachment to Optically Trapped Beads in Microstructures Monitored byBead Displacement,” Bioimaging 6: 25-32 (1998). They then diffuse ashort distance before becoming immobilized on a surface for detection.However, most exonucleases exhibit sequence- and structure-dependentcleavage rates, resulting in difficulties in data analysis and matchingsets from partial sequences. In addition, ways to identify the bases onthe detection surface still have to be developed or improved.

Regardless of the detection system, methods which utilize exonucleaseshave not been developed into methods that meet today's demand for rapid,high-throughput sequencing. In addition, most exonucleases haverelatively slow turnover rates, and the proposed methods requireextensive pretreatment, labeling and subsequent immobilization of thetemplate DNA on the bead in the flowing stream of fluid, all of whichmake a realization into a simple high-throughput system morecomplicated.

Other, more direct approaches to DNA sequencing have been attempted,such as determining the spatial sequence of fixed and stretched DNAmolecules by scanned atomic probe microscopy. Problems encountered withusing these methods consist in the narrow spacing of the bases in theDNA molecule (only 0.34 nm) and their small physicochemical differencesto be recognized by these methods. See Hansma et al., “ReproducibleImaging and Dissection of Plasmid DNA Under Liquid with the Atomic ForceMicroscope,” Science 256: 1180-1184 (1992).

In a recent approach for microsequencing using polymerase, but notexonuclease, a set of identical single stranded DNA (ssDNA) moleculesare linked to a substrate and the sequence is determined by repeating aseries of reactions using fluorescently labelled dNTPs. U.S. Pat. No.5,302,509 to Cheeseman. However, this method requires that each base isadded with a fluorescent label and 3′-dNTP blocking groups. After thebase is added and detected, the fluorescent label and the blocking groupare removed, and, then, the next base is added to the polymer.

Thus, the current sequencing methods either require both polymerase andexonuclease activity to deduce the sequence or rely on polymerase alonewith additional steps of adding and removing 3′-blocked dNTPs. The humangenome project has intensified the demand for rapid, small- andlarge-scale DNA sequencing that will allow high throughput with minimalstarting material. There also remains a need to provide a method forsequencing nucleic acid molecules that requires only polymeraseactivity, without the use of blocking substituents, resulting in greatersimplicity, easier miniaturizability, and compatibility to parallelprocessing of a single-step technique.

The present invention is directed to meeting the needs and overcomingdeficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a method of sequencing a target nucleicacid molecule having a plurality of nucleotide bases. This methodinvolves providing a complex of a nucleic acid polymerizing enzyme andthe target nucleic acid molecule oriented with respect to each other ina position suitable to add a nucleotide analog at an active sitecomplementary to the target nucleic acid. A plurality of types ofnucleotide analogs are provided proximate to the active site, whereineach type of nucleotide analog is complementary to a differentnucleotide in the target nucleic acid sequence. A nucleotide analog ispolymerized at an active site, wherein the nucleotide analog being addedis complementary to the nucleotide of the target nucleic acid, leavingthe added nucleotide analog ready for subsequent addition of nucleotideanalogs. The nucleotide analog added at the active site as a result ofthe polymerizing step is identified. The steps of providing a pluralityof nucleotide analogs, polymerizing, and identifying are repeated sothat the sequence of the target nucleic acid is determined.

Another aspect of the present invention relates to an apparatus suitablefor sequencing a target nucleic acid molecule. This apparatus includes asupport as well as a nucleic acid polymerizing enzyme or oligonucleotideprimer suitable to bind to a target nucleic acid molecule, where thepolymerase or oligonucleotide primer is positioned on the support. Amicrostructure defines a confined region containing the support and thenucleic acid polymerizing enzyme or the oligonucleotide primer which isconfigured to permit labeled nucleotide analogs that are not positionedon the support to move rapidly through the confined region.

A further feature of the present invention involves an apparatussuitable for sequencing a target nucleic acid molecule. This apparatusincludes a solid support and a nucleic acid polymerizing enzyme oroligonucleotide primer suitable to hybridize to a target nucleic acidmolecule, where the nucleic acid polymerizing enzyme or oligonucleotideprimer is positioned on the support. A housing defines a confined regioncontaining the support and the nucleic acid polymerizing enzyme or theoligonucleotide primer. The housing is constructed to facilitateidentification of labeled nucleotide analogs positioned on the support.Optical waveguides proximate to the confined region focus activatingradiation on the confined region and collect radiation from the confinedregion.

Numerous advantages are achieved with the present invention. Sequencingcan be carried out with small amounts of nucleic acid, with thecapability of sequencing single nucleic acid template molecules whicheliminates the need for amplification prior to initiation of sequencing.Long read lengths of sequence can be deduced in one run, eliminating theneed for extensive computational methods to assemble a gap-free fulllength sequence of long template molecules (e.g., bacterial artificialchromosome (BAC) clones). For two operational modes of the presentinventions, the read length of the sequence is limited by the length oftemplate to be sequenced, or the processivity of the polymerase,respectively. By using the appropriate enzymatic systems, e.g. withaccessory proteins to initiate the sequencing reaction at specific sites(e.g., origins of replication) on the double-stranded template nucleicacid, preparative steps necessary for conventional sequencingtechniques, such as subcloning into sequencing vectors, can beeliminated.

In addition, the sequencing method of the present invention can becarried out using polymerase and no exonuclease. This results in greatersimplicity, easier miniaturizability, and compatibility to parallelprocessing of a single-step technique.

In regard to the latter advantage, some polymerases exhibit higherprocessivity and catalytic speeds than exonucleases, with over 10,000bases being added before dissociation of the enzyme for the case of T7DNA polymerase (compared to 3,000 bases for λ exonuclease). In somecases, e.g., T7 DNA polymerase complexed with T7 helicase/primase,processivity values are even higher, ranging into several 100,000s. Therates of DNA synthesis can be very high, measured in vivo of 1,000bases/sec and in vitro of 750 bases/sec (in contrast to 12 bases/secdegraded by λ exonuclease in vitro). See Kelman et al., “Processivity ofDNA Polymerases: Two Mechanisms, One Goal,” Structure 6: 121-125 (1998);Carter et al., “The Role of Exonuclease and Beta Protein of Phage Lambdain Genetic Recombination. II. Substrate Specificity and the Mode ofAction of Lambda Exonuclease,” J. Biol. Chem. 246: 2502-2512 (1971);Tabor et al., “Escherichia coli Thioredoxin Confers Processivity on theDNA Polymerase Activity of the Gene 5 Protein of Bacteriophage T7,” J.Biol. Chem. 262: 16212-16223 (1987); and Kovall et al., “ToroidalStructure of Lambda-Exonuclease” Science 277: 1824-1827 (1997), whichare hereby incorporated by reference. An incorporation rate of 750bases/sec is approximately 150 times faster than the sequencing speed ofone of the fully automated ABI PRISM 3700 DNA sequencers by Perkin ElmerCorp., Foster City, Calif., proposed to be utilized in a shot-gunsequencing strategy for the human genome. See Venter et al., “ShotgunSequencing of the Human Genome,” Science 280: 1540-1542 (1998), which ishereby incorporated by reference.

The small size of the apparatus that can be used to carry out thesequencing method of the present invention is also highly advantageous.The confined region of the template/polymerase complex can be providedby the microstructure apparatus with the possibility of arrays enablinga highly parallel operational mode, with thousands of sequencingreactions carried out sequentially or simultaneously. This provides afast and ultrasensitive tool for research application as well as inmedical diagnostics.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-C show 3 alternative embodiments for sequencing in accordancewith the present invention.

FIGS. 2A-C are schematic drawings showing the succession of steps usedto sequence nucleic acids in accordance with the present invention.

FIGS. 3A-C show plots of fluorescence signals vs. time during thesuccession of steps used to sequence the nucleic acid in accordance withthe present invention. FIG. 3C shows the sequence generated by thesesteps.

FIGS. 4A-D depict the structure and schematic drawings showing thesuccession of steps used to sequence the nucleic acid in accordance withthe present invention in the case where fluorescent nucleotides carryingthe label at the gamma phosphate position (here shown as a gamma-linkeddNTP) are used.

FIG. 5 shows the principle of discrimination of fluorophores bytime-gated fluorescence decay time measurements, which can be used tosuppress background signal in accordance with the present invention.

FIG. 6A shows a system for sequencing in accordance with the presentinvention. FIG. 6B is an enlargement of a portion of that system.

FIG. 7A shows a system for sequencing in accordance with the presentinvention using electromagnetic field enhancement with metal tips. FIG.7B is an enlargement of a portion of that system.

FIG. 8A shows a system for sequencing in accordance with the presentinvention using near field apertures. FIG. 8B is an enlargement of aportion of that system.

FIG. 9A shows a system for sequencing in accordance with the presentinvention using nanochannels. FIG. 9B is an enlargement of a portion ofthat system.

FIGS. 10A-B show systems for supplying reagents to a nanofabricatedconfinement system in accordance with the present invention. Inparticular, FIG. 10A is a schematic drawing which shows how reagents areprovided and passed through the system. FIG. 10B is similar but showsthis system on a single chip with pads to connect the system to fluidreservoirs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of sequencing a target nucleicacid molecule having a plurality of nucleotide bases. This methodinvolves providing a complex of a nucleic acid polymerizing enzyme andthe target nucleic acid molecule oriented with respect to each other ina position suitable to add a nucleotide analog at an active sitecomplementary to the target nucleic acid. A plurality of types ofnucleotide analogs are provided proximate to the active site, whereineach type of nucleotide analog is complementary to a differentnucleotide in the target nucleic acid sequence. A nucleotide analog ispolymerized at an active site, wherein the nucleotide analog being addedis complementary to the nucleotide of the target nucleic acid, leavingthe added nucleotide analog ready for subsequent addition of nucleotideanalogs. The nucleotide analog added at the active site as a result ofthe polymerizing step is identified. The steps of providing a pluralityof nucleotide analogs, polymerizing, and identifying are repeated sothat the sequence of the target nucleic acid is determined.

Another aspect of the present invention relates to an apparatus suitablefor sequencing a target nucleic acid molecule. This apparatus includes asupport as well as a nucleic acid polymerizing enzyme or oligonucleotideprimer suitable to bind to a target nucleic acid molecule, where thepolymerase or oligonucleotide primer is positioned on the support. Amicrostructure defines a confined region containing the support and thenucleic acid polymerizing enzyme or the oligonucleotide primer which isconfigured to permit labeled nucleotide analogs that are not positionedon the support to move rapidly through the confined region.

A further feature of the present invention involves an apparatussuitable for sequencing a target nucleic acid molecule. This apparatusincludes a support and a nucleic acid polymerizing enzyme oroligonucleotide primer suitable to hybridize to a target nucleic acidmolecule, where the nucleic acid polymerizing enzyme or oligonucleotideprimer is positioned on the support. A housing defines a confined regioncontaining the support and the nucleic acid polymerizing enzyme or theoligonucleotide primer. The housing is constructed to facilitateidentification of labeled nucleotide analogs positioned on the support.Optical waveguides proximate to the confined region focus activatingradiation on the confined region and collect radiation from the confinedregion.

The present invention is directed to a method of sequencing a targetnucleic acid molecule having a plurality of bases. In its fundamentalprinciple, the temporal order of base additions during thepolymerization reaction is measured on a single molecule of nucleicacid, i.e. the activity of a nucleic acid polymerizing enzyme, hereafteralso referred to as polymerase, on the template nucleic acid molecule tobe sequenced is followed in real time. The sequence is deduced byidentifying which base is being incorporated into the growingcomplementary strand of the target nucleic acid by the catalyticactivity of the nucleic acid polymerizing enzyme at each step in thesequence of base additions. In the preferred embodiment of theinvention, recognition of the time sequence of base additions isachieved by detecting fluorescence from appropriately labellednucleotide analogs as they are incorporated into the growing nucleicacid strand. Accuracy of base pairing is provided by the specificity ofthe enzyme, with error rates of false base pairing of 10⁻⁵ or less. Forenzyme fidelity, see Johnson, “Conformational Coupling in DNA-PolymeraseFidelity,” Ann. Rev. Biochem. 62:685-713 (1993) and Kunkel,“DNA-Replication Fidelity,” J. Biol. Chem. 267:18251-18254 (1992), whichare hereby incorporated by reference.

The invention applies equally to sequencing all types of nucleic acids(DNA, RNA, DNA/RNA hybrids etc.) using a number of polymerizing enzymes(DNA polymerases, RNA polymerases, reverse transcriptases, mixtures,etc.). Therefore, appropriate nucleotide analogs serving as substratemolecules for the nucleic acid polymerizing enzyme can consist ofmembers of the groups of dNTPs, NTPs, modified dNTPs or NTPs, peptidenucleotides, modified peptide nucleotides, or modified phosphate-sugarbackbone nucleotides.

There are two convenient operational modes in accordance with thepresent invention. In the first operational mode of the invention, thetemplate nucleic acid is attached to a support. This can be either byimmobilization of (1) an oligonucleotide primer or (2) a single-strandedor (3) double-stranded target nucleic acid molecule. Then, either (1)the target nucleic acid molecule is hybridized to the attachedoligonucleotide primer, (2) an oligonucleotide primer is hybridized tothe immobilized target nucleic acid molecule, to form a primed targetnucleic acid molecule complex, or (3) a recognition site for thepolymerase is created on the double stranded template (e.g., throughinteraction with accessory proteins, such as a primase). A nucleic acidpolymerizing enzyme on the primed target nucleic acid molecule complexis provided in a position suitable to move along the target nucleic acidmolecule and extend the oligonucleotide primer at an active site. Aplurality of labelled types of nucleotide analogs, which do not have ablocking substituent, are provided proximate to the active site, witheach distinguishable type of nucleotide analog being complementary to adifferent nucleotide in the target nucleic acid sequence. Theoligonucleotide primer is extended by using the nucleic acidpolymerizing enzyme to add a nucleotide analog to the oligonucleotideprimer at the active site, where the nucleotide analog being added iscomplementary to the nucleotide of the target nucleic acid at the activesite. The nucleotide analog added to the oligonucleotide primer as aresult of the extending step is identified. If necessary, the labelednucleotide analog, which is added to the oligonucleotide primer, istreated before many further nucleotide analogs are incorporated into theoligonucleotide primer to insure that the nucleotide analog added to theoligonucleotide primer does not prevent detection of nucleotide analogsin subsequent polymerization and identifying steps. The steps ofproviding labelled nucleotide analogs, extending the oligonucleotideprimer, identifying the added nucleotide analog, and treating thenucleotide analog are repeated so that the oligonucleotide primer isfurther extended and the sequence of the target nucleic acid isdetermined.

Alternatively, the above-described procedure can be carried out by firstattaching the nucleic acid polymerizing enzyme to a support in aposition suitable for the target nucleic acid molecule complex to moverelative to the nucleic acid polymerizing enzyme so that the primednucleic acid molecular complex is extended at an active site. In thisembodiment, a plurality of labelled nucleotide analogs complementary tothe nucleotide of the target nucleic acid at the active site are addedas the primed target nucleic acid complex moves along the nucleic acidpolymerizing enzyme. The steps of providing nucleotide analogs,extending the primer, identifying the added nucleotide analog, andtreating the nucleotide analog during or after incorporation arerepeated, as described above, so that the oligonucleotide primer isfurther extended and the sequence of the target nucleic acid isdetermined.

FIGS. 1A-C show 3 alternative embodiments for sequencing in accordancewith the present invention. In FIG. 1A, a sequencing primer is attachedto a support, e.g. by a biotin-streptavidin bond, with the primerhybridized to the target nucleic acid molecule and the nucleic acidpolymerizing enzyme attached to the hybridized nucleic acid molecule atthe active site where nucleotide analogs are being added to thesequencing primer. In FIG. 1B, the target nucleic acid molecule isattached to a support, with a sequencing primer hybridized to thetemplate nucleic acid molecule and the nucleic acid polymerizing enzymeattached to the hybridized nucleic molecule at the active site wherenucleotide analogs are being added to the sequencing primer. The primercan be added before or during the providing of nucleotide analogs. Inaddition to these scenarios, a double stranded target nucleic acidmolecule can be attached to a support, with the target nucleic acidmolecule harboring a recognition site for binding of the nucleic acidpolymerizing enzyme at an active site where nucleotide analogs are beingadded to the primer. For example, such a recognition site can beestablished with the help of an accessory protein, such as an RNApolymerase or a helicase/primase, which will synthesize a short primerat specific sites on the target nucleic acid and thus provide a startingsite for the nucleic acid polymerizing enzyme. See Richardson“Bacteriophage T7: Minimal Requirements for the Replication of a DuplexDNA Molecule,” Cell 33: 315-317 (1983), which is hereby incorporated byreference. In FIG. 1C, the nucleic acid polymerizing enzyme is attachedto a support, with the primed target nucleic acid molecule binding atthe active site where nucleotide analogs are being added to thesequencing primer. As in the previous description, the nucleic acidpolymerizing enzyme can likewise be attached to a support, but with thetarget nucleic acid molecule being double-stranded nucleic acid,harboring a recognition site for binding of the nucleic acidpolymerizing enzyme at an active site where nucleotide analogs are beingadded to the growing nucleic acid strand. Although FIGS. 1A-C show onlyone sequencing reaction being carried out on the support, it is possibleto conduct an array of several such reactions at different sites on asingle support. In this alternative embodiment, each sequencing primer,target nucleic acid, or nucleic acid polymerizing enzyme to beimmobilized on this solid support is spotted on that surface bymicrocontact printing or stamping, e.g., as is used for microarraytechnology of DNA chips, or by forming an array of binding sites bytreating the surface of the solid support. It is also conceivable tocombine the embodiments outlined in FIG. 1 and immobilize both thetarget nucleic acid molecule and the nucleic acid polymerizing enzymeproximate to each other.

The sequencing process of the present invention can be used to determinethe sequence of any nucleic acid molecule, including double-stranded orsingle-stranded DNA, single stranded DNA hairpins, DNA/RNA hybrids, RNAwith a recognition site for binding of the polymerase, or RNA hairpins.

The sequencing primer used in carrying out the process of the presentinvention can be a ribonucleotide, deoxyribonucleotide, modifiedribonucleotide, modified deoxyribonucleotide, peptide nucleic acid,modified peptide nucleic acid modified phosphate-sugar backboneoligonucleotide, and other nucleotide and oligonucleotide analogs. Itcan be either synthetic or produced naturally by primases, RNApolymerases, or other oligonucleotide synthesizing enzymes.

The nucleic acid polymerizing enzyme utilized in accordance with thepresent invention can be either a thermostable polymerase or a thermallydegradable polymerase. Examples for suitable thermostable polymerasesinclude polymerases isolated from Thermus aquaticus, Thermusthermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcuslitoralis, and Thermotoga maritima. Useful thermodegradable polymersasesinclude E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, T4 DNA polymerase, T7 DNA polymerase, and others. Examplesfor other polymerizing enzymes that can be used to determine thesequence of nucleic acid molecules include E. coli, T7, T3, SP6 RNApolymerases and AMV, M-MLV and HIV reverse transcriptases. Thepolymerase can be bound to the primed target nucleic acid sequence at aprimed single-stranded nucleic acid, an origin of replication, a nick orgap in a double-stranded nucleic acid, a secondary structure in asingle-stranded nucleic acid, a binding site created by an accessoryprotein, or a primed single-stranded nucleic acid.

Materials which are useful in forming the support include glass, glasswith surface modifications, silicon, metals, semiconductors, highrefractive index dielectrics, crystals, gels, and polymers.

In the embodiments of FIG. 1, any suitable binding partner known tothose skilled in the art could be used to immobilize either thesequencing primer, the target nucleic acid molecule, or the nucleic acidpolymerizing enzyme to the support. Non-specific binding by adsorptionis also possible. As shown in FIGS. 1A-C, a biotin-streptavidin linkageis suitable for binding the sequencing primer or the target nucleic acidmolecule to the solid support. The biotin component of such a linkagecan be attached to either the primer or nucleic acid or to the solidsupport with the streptavidin (or any other biotin-binding protein)being attached to the opposite entity.

One approach for carrying out this binding technique involves attachingPHOTOACTIVATABLE BIOTIN™ (“PAB”) (Pierce Chemical Co., Rockford, Ill.)to a surface of the chamber used to carry out the sequencing procedureof the present invention. This can be achieved by exposure to light at360 nm, preferably through a transparent wall of the chamber, asdescribed in Hengsakul et al., “Protein Patterning with a PhotoactivableDerivative of Biotin,” Bioconjugate Chem. 7: 249-54 (1996), which ishereby incorporated by reference. When using a nanochamber, the biotinis activated in a diffraction-limited spot under an optical microscope.With near-field excitation, exposure can be self-aligned using awaveguide to direct light to the desired area. When exposed to light thePAB is activated and binds covalently to the interior surface of thechannel. Excess unbound PAB is then removed by flushing with water.

Alternatively, streptavidin can be coated on the support surface. Theappropriate nucleic acid primer oligonucleotide or the single strandednucleic acid template is then biotinylated, creating an immobilizednucleic acid primer-target molecule complex by virtue of thestreptavidin-biotin bound primer.

Another approach for carrying out the process of the present inventionis to utilize complementary nucleic acids to link the sequencing primeror the target nucleic acid molecule to the solid support. This can becarried out by modifying a single stranded nucleic acid with a knownleader sequence and ligating the known leader sequence to the sequencingprimer or the target nucleic acid molecule. The resultingoligonucleotide may then be bound by hybridization to an oligonucleotideattached to the support and having a nucleotide sequence complementaryto that of the known leader sequence. Alternatively, a secondoligonucleotide can be hybridized to an end of the target nucleic acidmolecule opposite to that bound to the oligonucleotide primer. Thatsecond oligonucleotide is available for hybridization to a complementarynucleic sequence attached to the support.

Reversible or irreversible binding between the support and either theoligonucleotide primer or the target nucleic acid sequence can beachieved with the components of any covalent or non-covalent bindingpair. Other such approaches for immobilizing the sequencing primer orthe target nucleic acid molecule to the support include anantibody-antigen binding pair and photoactivated coupling molecules.

In the embodiment of FIG. 1C, any technique known to be useful inreversibly or irreversibly immobilizing proteinaceous materials can beemployed. It has been reported in the literature that RNA polymerase wassuccessfully immobilized on activated surfaces without loss of catalyticactivity. See Yin et al., “Transcription Against an Applied Force,”Science 270:1653-1657 (1995), which is hereby incorporated by reference.Alternatively, the protein can be bound to an antibody, which does notinterfere with its catalytic activity, as has been reported for HIVreverse transcriptase. See Lennerstrand et al., “A Method for CombinedImmunoaffinity Purification and Assay of HIV-1 Reverse TranscriptaseActivity Useful for Crude Samples,” Anal. Biochem. 235:141-152 (1996),which is hereby incorporated by reference. Therefore, nucleic acidpolymerizing enzymes can be immobilized without loss of function. Theantibodies and other proteins can be patterned on inorganic surfaces.See James et al., “Patterned Protein Layers on Solid Substrates by ThinStamp Microcontact Printing,” Langmuir 14:741-744 (1998) and St John etal., “Diffraction-Based Cell Detection Using a Microcontact PrintedAntibody Grating,” Anal. Chem. 70:1108-1111 (1998), which are herebyincorporated by reference. Alternatively, the protein could bebiotinylated (or labelled similarly with other binding molecules), andthen bound to a streptavidin-coated support surface.

In any of the embodiments of FIGS. 1A to C, the binding partner andeither the polymerase or nucleic acids they immobilize can be applied tothe support by conventional chemical and photolithographic techniqueswhich are well known in the art. Generally, these procedures can involvestandard chemical surface modifications of the support, incubation ofthe support at different temperatures in different media, and possiblesubsequent steps of washing and incubation of the support surface withthe respective molecules.

Alternative possibilities of positioning of the polymerizing complex areconceivable, such as by entrapment of the complex in a gel harboringpores too small to allow passage of the complex, but large enough toaccommodate delivery of nucleotide analogs. Suitable media includeagarose gels, polyacrylamide gels, synthetic porous materials, ornanostructures.

The sequencing procedure of the present invention can be initiated byaddition of nucleic acid polymerizing enzyme to the reaction mixture inthe embodiment of FIGS. 1A-B. For the embodiment of FIG. 1C, the primednucleic acid can be added for initiation. Other scenarios for initiationcan be employed, such as establishing a preformed nucleicacid-polymerase complex in the absence of divalent metal ions which areintegral parts of the active sites of polymerases (most commonly Mg²⁺).The sequencing reaction can then be started by adding these metal ions.The preinitiation complex of template could also be formed with theenzyme in the absence of nucleotides, with fluorescent nucleotideanalogs being added to start the reaction. See Huber et al.,“Escherichia coli Thioredoxin Stabilizes Complexes of Bacteriophage T7DNA Polymerase and Primed Templates,” J. Biol. Chem. 262:16224-16232(1987), which is hereby incorporated by reference. Alternatively, theprocess can be started by uncaging of a group on the oligonucleotideprimer which protects it from binding to the nucleic acid polymerizingenzyme. Laser beam illumination would then start the reactioncoincidentally with the starting point of observation.

FIGS. 2A-C are schematic drawings showing the succession of steps usedto sequence nucleic acids in accordance with the present invention.

In FIG. 2A, labelled nucleotide analogs are present in the proximity ofthe primed complex of a nucleic acid polymerizing enzyme attached to thehybridized sequencing primer and target nucleic acid molecule which areattached on the solid support. During this phase of the sequencingprocess, the labelled nucleotide analogs diffuse or are forced to flowthrough the extension medium towards and around the primed complex.

In accordance with FIG. 2B, once a nucleotide analog has reached theactive site of the primed complex, it is bound to it and the nucleicacid polymerizing enzyme establishes whether this nucleotide analog iscomplementary to the first open base of the target nucleic acid moleculeor whether it represents a mismatch. The mismatched base will berejected with the high probability that corresponds to theabove-mentioned high fidelity of the enzyme, whereas the complementarynucleotide analog is polymerized to the sequencing primer to extend thesequencing primer.

During or after each labelled nucleotide analog is added to thesequencing primer, the nucleotide analog added to the sequencing primeris identified. This is most efficiently achieved by giving eachnucleotide analog a different distinguishable label. By detecting whichof the different labels are added to the sequencing primer, thecorresponding nucleotide analog added to the sequencing primer can beidentified and, by virtue of its complementary nature, the base of thetarget nucleic acid which the nucleotide analog complements can bedetermined. Once this is achieved, it is no longer necessary for thenucleotide analog that was added to the sequencing primer to retain itslabel. In fact, the continued presence of labels on nucleotide analogscomplementing bases in the target nucleic acid that have already beensequenced would very likely interfere with the detection of nucleotideanalogs subsequently added to the primer. Accordingly, labels added tothe sequencing primer are removed after they have been detected, asshown in FIG. 2C. This preferably takes place before additionalnucleotide analogs are incorporated into the oligonucleotide primer.

By repeating the sequence of steps described in FIGS. 2A-C, thesequencing primer is extended and, as a result, the entire sequence ofthe target nucleic acid can be determined. Although the immobilizationembodiment depicted in FIGS. 2A-C is that shown in FIG. 1A, thealternative immobilization embodiments shown in FIGS. 1B-C couldsimilarly be utilized in carrying out the succession of steps shown inFIGS. 2A-C.

In carrying out the diffusion, incorporation, and removal steps of FIGS.2A-C, an extension medium containing the appropriate components topermit the nucleotide analogs to be added to the sequencing primer isused. Suitable extension media include, e.g., a solution containing 50mM Tris-HCl, pH 8.0, 25 mM MgCl₂, 65 mM NaCl, 3 mM DTT, (this is theextension medium composition recommended by the manufacturer forSequenase, a T7 mutant DNA polymerase), and nucleotide analogs at anappropriate concentration to permit the identification of the sequence.Other media that are appropriate for this and other polymerases arepossible, with or without accessory proteins, such as single-strandedbinding proteins. Preferably, the extension phase is carried out at 37°C. for most thermally degradable polymerases, although othertemperatures at which the polymerase is active can be employed.

Once a labelled nucleotide analog is added to the sequencing primer, asnoted above, the particular label of the added moiety must be identifiedin order to determine which type of nucleotide analog was added to thesequencing primer and, as a result, what the complementary base oftarget nucleic acid is. How the label of the added entity is determineddepends upon the type of label being utilized. For the preferredembodiment of the invention, discussion of the identification steps willbe restricted to the employment of nucleotide analogs carryingfluorescent moieties. However, other suitable labels includechromophores, enzymes, antigens, heavy metals, magnetic probes, dyes,phosphorescent groups, radioactive materials, chemiluminescent moieties,scattering or fluorescent nanoparticles, Raman signal generatingmoieties, and electrochemical detecting moieties. Such labels are knownin the art and are disclosed for example in Prober, et. al., Science238: 336-41 (1997); Connell et. al., BioTechniques 5(4): 342-84 (1987);Ansorge, et. al., Nucleic Acids Res. 15(11): 4593-602 (1987); and Smithet. al., Nature 321:674 (1986), which are hereby incorporated byreference. In some cases, such as for chromophores, fluorophores,phosphorescent labels, nanoparticles, or Raman signaling groups, it isnecessary to subject the reaction site to activating radiation in orderto detect the label. This procedure will be discussed in detail belowfor the case of fluorescent labels. Suitable techniques for detectingthe fluorescent label include time-resolved far-field microspectroscopy,near-field microspectroscopy, measurement of fluorescence resonanceenergy transfer, photoconversion, and measurement of fluorescencelifetimes. Fluorophore identification can be achieved by spectralwavelength discrimination, measurement and separation of fluorescencelifetimes, fluorophore identification, and/or background suppression.Fluorophore identification and/or background suppression can befacilitated by fast switching between excitation modes and illuminationsources, and combinations thereof.

FIGS. 3A-B show plots of fluorescence signals vs. time during thesuccession of steps (outlined in FIG. 2) that is used to carry out thesequencing procedure of the present invention. In essence, in thisprocedure, an incorporated nucleotide analog will be distinguished fromunincorporated ones (randomly diffusing through the volume ofobservation or being convected through it by hydrodynamic orelectrophoretic flow) by analyzing the time trace of fluorescence foreach distinguishable label simultaneously. This is achieved by photonburst recordings and time-resolved fluorescence correlation spectroscopywhich distinguishes the continuing steady fluorescence of theincorporated label (until removed by the mechanisms discussed below)from the intennittent emission of the free fluorophores. See Magde etal., “Thermodynamic Fluctuations in a Reacting System—Measurement byFluorescence Correlation Spectroscopy,” Phys. Rev. Lett. 29:705-708(1972), Kask P. et al., “Fluorescence-Intensity Distribution Analysisand its Application in Biomolecular Detection Technology,” Proc. Nat.Acad. Sci. U.S.A. 96: 13756-13761 (1999), and Eggeling et al.,“Monitoring Conformational Dynamics of a Single Molecule by SelectiveFluorescence Spectroscopy,” Proc. Nat. Acad. Sci. U.S.A. 95: 1556-1561(1998), which are hereby incorporated by reference. The sequence can bededuced by combining time traces of all detection channels.

FIG. 3A shows a plot of fluorescence signal vs. time during just thediffusion phase of FIG. 2A, assuming four different channels offluorescence detection for the four different bases (e.g., by employingfour different labels, each with a different fluorescence emissionspectrum, by which they can be separated through optical filters). Eachpeak in FIG. 3A represents the burst of fluorescence resulting from thepresence of a nucleotide analog in the volume of observation, with eachdifferent nucleotide analog being distinguished by its different labelwhich generates peaks of different colors (depicted in FIG. 3A bydifferent line patterns). The narrow width of these peaks indicates thatthe nucleotide analogs have a brief residence time proximate to theactive site of sequencing, because they are freely diffusing or flowingthrough the volume of observation. A peak of similar width is expectedfor the case of a mismatched nucleotide analog transiently binding tothe active site of the nucleic acid polymerizing enzyme, and subsequentrejection of incorporation by the enzyme.

FIG. 3B shows a plot of fluorescence signal vs. time during theincorporation and subsequent removal phases of FIGS. 2B-C. As in FIG.3A, each peak of FIG. 3B represents the presence of a nucleotide analogwith each different nucleotide analog being distinguished by itsdifferent label which generates peaks of different colors (depicted inFIG. 3B by different line patterns). The narrow width of some peaks inFIG. 3B again relates to the nucleotide analogs which remain mobilewithin the extension medium and do not extend the sequencing primer.Such narrow peaks result because these nucleotide analogs have a briefresidence time proximate to the active site of sequencing, as explainedfor FIG. 3A. On the other hand, the wider peaks correspond to nucleotideanalogs which have, at the active site, complementary bases on thetemplate nucleic acid molecule and serve to extend the sequencingprimer. As a result of their immobilization, these nucleotide analogshave wider peaks, because they will remain in the observation volumeduring and after incorporation in the growing nucleic acid strand, andthus continue to emit fluorescence. Their signal is only terminatedlater in time as a result of the subsequent removal step whicheliminates continued fluorescence, and allowing the identification ofsubsequent incorporation events.

Moving from left to right in FIG. 3B (i.e. later in time), the sequenceof wider peaks corresponds to the complement of the sequence of thetemplate nucleic acid molecule. FIG. 3C shows the final output of FIG.3B which can be achieved, for example, by a computer program thatdetects the short bursts of fluorescence and discards them in the finaloutput. As a result of such filtering, only the peaks generated byimmobilized nucleotide analogs are present, and converted into thesequence corresponding to the complement of sequence of the templatenucleic acid molecule. This complementary sequence is here ATACTA,therefore, the order of the bases of the template nucleic acid moleculebeing sequenced is TATGAT.

Fluorescent labels can be attached to nucleotides at a variety oflocations. Attachment can be made either with or without a bridginglinker to the nucleotide. Conventionally used nucleotide analogs forlabeling of nucleic acid with fluorophores carry the fluorescent moietyattached to the base of the nucleotide substrate molecule. However, itcan also be attached to a sugar moiety (e.g., deoxyribose) or the alphaphosphate. Attachment to the alpha phosphate might prove advantageous,because this kind of linkage leaves the internal structure of thenucleic acid intact, whereas fluorophores attached to the base have beenobserved to distort the double helix of the synthesized molecule andsubsequently inhibit further polymerase activity. See Zhu et al.,“Directly Labelled DNA Probes Using Fluorescent Nucleotides withDifferent Length Linkers,” Nucleic Acids Res. 22: 3418-3422 (1994), andDoublie et al., “Crystal Structure of a Bacteriophage T7 DNA ReplicationComplex at 2.2 Ångstrom Resolution,” Nature 391:251-258 (1998), whichare hereby incorporated by reference. Thus, thiol-group-containingnucleotides, which have been used (in the form of NTPs) forcross-linking studies on RNA polymerase, could serve as primary backbonemolecules for the attachment of suitable linkers and fluorescent labels.See Hanna et al., “Synthesis and Characterization of a NewPhoto-Cross-Linking CTP Analog and Its Use in Photoaffinity-LabelingEscherichia-coli and T7-RNA Polymerases,” Nucleic Acids Res.21:2073-2079 (1993), which is hereby incorporated by reference.

In the conventional case where the fluorophore is attached to the baseof the nucleotide, it is typically equipped with fluorophores of arelatively large size, such as fluorescein. However, smallerfluorophores, e.g., pyrene or dyes from the coumarin family, could proveadvantageous in terms of being tolerated to a larger extent bypolymerases. In fact, it is possible to synthesize a DNA fragment of7,300 base pair length in which one base type is fully replaced by thecorresponding coumarin-labelled dNTP using T7 DNA polymerase, whereasthe enzyme is not able to carry out the corresponding synthesis usingfluorescein-labelled dNTPs.

In all of these cases, the fluorophore remains attached to the part ofthe substrate molecule that is incorporated into the growing nucleicacid molecule during synthesis. Suitable means for removal of thefluorophore after it has been detected and identified in accordance withthe sequencing scheme of the present invention include photobleaching ofthe fluorophore or photochemical cleavage of the nucleotide and thefluorophore, e.g., cleavage of a chemical bond in the linker. Removal ofthe fluorescent label of already incorporated nucleotides, the rate ofwhich can be adjusted by the laser power, prevents accumulation ofsignal on the nucleic acid strand, thereby maximizing the signal tobackground ratio for nucleotide identification. For this scheme, theobjective of the present invention is to detect all of the photons fromeach label and then photobleach or photochemically cleave before or soonafter the next few nucleotide is incorporated in order to maintainadequate signal to noise values for subsequent identification steps. Theremoval phase of the process of the present invention can be carried outby any procedure suitable for removing a label without damaging thesequencing reaction complex.

In addition to fluorescent labels that remain in the nucleic acid duringsynthesis, nucleotides that are labelled fluorescently or otherwise andcarry the label attached to either the beta or gamma phosphate of thenucleotide can also be used in the sequencing procedure of the presentinvention. Analogous compounds have previously been synthesized in theform of NTP analogs and have been shown to be excellent substrates for avariety of enzymes, including RNA polymerases. See Yarbrough et al.,“Synthesis and Properties of Fluorescent Nucleotide Substrates forDNA-dependent RNA Polymerase,” Journal of Biological Chemistry254:12069-12073 (1979), and Chatterji et al., “Fluorescence SpectroscopyAnalysis of Active and Regulatory Sites of RNA Polymerase,” Methods inEnzymology 274: 456-479 (1996), which are hereby incorporated byreference. During the synthesis of DNA, the bond cleavage in thenucleotide occurs between the alpha and the beta phosphate, causing thebeta and gamma phosphates to be released from the active site afterpolymerization, and the formed pyrophosphate subsequently diffuses or isconvected away from the nucleic acid. In accordance with the presentinvention, it is possible to distinguish the event of binding of anucleotide and its incorporation into nucleic acid from events justinvolving the binding (and subsequent rejection) of a mismatchednucleotide, because the rate constants of these two events aredrastically different. The rate-limiting step in the successiveelementary steps of DNA polymerization is a conformational change of thepolymerase that can only occur after the enzyme has established that thecorrect (matched) nucleotide is bound to the active site. Therefore, anevent of a mismatched binding of a nucleotide analog will be muchshorter in time than the event of incorporation of the correct base. SeePatel et al., “Pre-Steady-State Kinetic Analysis of Processive DNAReplication Including Complete Characterization of anExonuclease-Deficient Mutant,” Biochemistry 30: 511-525 (1991) and Wonget al., “An Induced-Fit Kinetic Mechanism for DNA Replication Fidelity:Direct Measurement by Single-Turnover Kinetics,” Biochemistry 30:511-525 (1991), which are hereby incorporated by reference. As a result,the fluorescence of the label that is attached to the beta or gammaphosphate of the nucleotide analog remains proximate to the polymerasefor a longer time in case the nucleotide analog is polymerized, and canbe distinguished in accordance to the scheme described above for FIG. 3.After incorporation, the label will diffuse away with the cleavedpyrophosphate. This procedure is shown in FIG. 4. FIG. 4A shows thestructure of 1-aminonaphthalene-5-sulfonate (AmNS)-dUTP, arepresentative example of a nucleotide analog carrying a fluorescentlabel attached to the gamma phosphate, with the cleavage positionindicated by the dashed line. FIG. 4B-D show the successive steps ofincorporation and release of the pyrophosphate-fluorophore complex, inanalogy to FIG. 2. The time trace of fluorescence for this scheme willbe the same as shown in FIG. 3. Thus, this is an alternative scheme tothe one outlined above in which the fluorophore is first incorporatedinto the nucleic acid and the signal is subsequently eliminated byphotobleaching or photochemical cleavage after identification of thelabel.

The identification of the particular fluorescently labelled nucleotideanalog that is incorporated against the background of unincorporatednucleotides diffusing or flowing proximally to the nucleic acidpolymerizing enzyme can be further enhanced by employing the observationthat for certain fluorescently labelled dNTPs (e.g., coumarin-5-dGTP, orAmNS-UTP), the presence of the base in the form of a covalent linkagesignificantly reduces (i.e. quenches) the fluorescence of the label. SeeDhar et al., “Synthesis and Characterization of Stacked and QuenchedUridine Nucleotide Fluorophores,” Journal of Biological Chemistry 274:14568-14572 (1999), and Draganescu et al., “Fhit-Nucleotide SpecificityProbed with Novel Fluorescent and Fluorogenic Substrates,” Journal ofBiological Chemistry 275: 4555-4560 (2000), which are herebyincorporated by reference. The interaction between the fluorophore andthe base quenches the fluorescence, so that the molecule is not veryfluorescent in solution by itself. However, when such a fluorescentnucleotide is incorporated into the nucleic acid, the fluorophore getsdisconnected from the nucleotide and the fluorescence is no longerquenched. For the case of a linkage to the beta or gamma phosphate ofthe nucleotide, this occurs naturally through the enzymatic activity ofthe polymerase, in the case of fluorophores linked to the base, thiswould have to be accomplished by photochemical cleavage. The signal offluorescence from the cleaved fluorophore is much brighter and can bedetected over the possible background of the plurality of quenchedmolecules in the vicinity of the polymerase/nucleic acid complex.

Furthermore, since the fluorescence lifetime of the quenched moleculesdiffusing in the solution is much shorter than the lifetime of thecleaved molecule, a further enhancement of signal to background can beachieved by employing pulsed illumination and time-gated photondetection. This is illustrated in FIG. 5, showing the time-resolvedfluorescence decay curves for coumarin alone and coumarin-dGTP,respectively. Because the coumarin fluorescence is quenched uponcovalent linkage to dGTP, the lifetime is much shorter than for the freedye alone, meaning that on average, fluorescent photons are emitted muchsooner after an excitation pulse, e.g., delivered by a pulsed laser. Byeliminating this time interval immediately after the pulse fromdetection, which can be achieved, for example, with a variable delayline component (indicated by the crosshatched bar with adjustable delaytime of width T), the response window of the detector can be gated suchthat only fluorescence emitted from the slow decay component, in thiscase the free dye (or, in tenns of the sequencing scheme, the cleavedfluorophore) is detected, and thus background from unincorporatedmolecules is reduced even further. Saavedra et al. “Time-ResolvedFluorimetric Detection of Terbium-Labelled Deoxyribonucleic AcidSeparated by Gel Electrophoresis,” Analyst 114:835-838 (1989), which ishereby incorporated by reference.

Nucleotides can also be converted into fluorophores by photochemicalreactions involving radical formation. This technique has been utilizedwith serotonin and other biologically relevant molecules. See Shear etal., “Multiphoton-Excited Visible Emission by Serotonin Solutions,”Photochem, Photobiol. 65:931-936 (1997), which is hereby incorporated byreference. The ideal photophysical situation would be to have eachnucleotide generate its own fluorescence signal. Unfortunately, nucleicacid and the individual nucleotides are poor fluorophores emittingweakly with minuscule quantum efficiencies and only on illumination withdeep ultraviolet light. However, the native ultraviolet fluorophoreserotonin (5HT) can be photoionized by simultaneous absorption of 4infrared photons, to form a radical that reacts with other ground statemolecules to form a complex that emits bright green fluorescence onabsorption of 2 more photons. Subsequent discoveries showed that manysmall organic molecules can undergo this multiphoton conversion.

Known quenching of fluorophores by nucleic acid components and byneighboring fluorophores as well as resonance energy transfer mayprovide markers tolerated by the polymerase. Furey et al., “Use ofFluorescence Resonance Energy Transfer to Investigate the Conformationof DNA Substrates Bound to the Klenow Fragment,” Biochemistry37:2979-2990 (1998) and Glazer et al., “Energy-Transfer FluorescentReagents for DNA Analyses,” Curr. Op. Biotechn. 8:94-102 (1997), whichare hereby incorporated by reference.

In the most efficient setup of the present invention, each base shouldbe distinguished by its own label so that the sequence can be deducedfrom the combined output of four different channels as illustrated inFIG. 3C. This can, for example, be accomplished by using differentfluorophores as labels and four different detection channels, separatedby optical filters. It is also possible to distinguish the labels byparameters other than the emission wavelength band, such as fluorescencelifetime, or any combination of several parameters for the differentbases. Due to the possible interactions of a fluorophore with a base, itis feasible to employ the same fluorophore to distinguish more than onebase. As an example, coumarin-dGTP has a much shorter fluorescencelifetime than coumarin-dCTP so that the two bases could be distinguishedby their difference in fluorescence lifetime in the identification stepof the sequencing scheme, although they carry the same chemicalsubstance as the fluorescent label.

The sequencing procedure can also be accomplished using less than 4labels employed. With 3 labels, the sequence can be deduced fromsequencing a nucleic acid strand (1) if the 4^(th) base can be detectedas a constant dark time delay between the signals of the other labels,or (2) unequivocally by sequencing both nucleic acid strands, because inthis case one obtains a positive fluorescence signal from each basepair. Another possible scheme that utilizes two labels is to have onebase labelled with one fluorophore and the other three bases withanother fluorophore. In this case, the other 3 bases do not give asequence, but merely a number of bases that occur between the particularbase being identified by the other fluorophore. By cycling thisidentifying fluorophore through the different bases in differentsequencing reactions, the entire sequence can be deduced from sequentialsequencing runs. Extending this scheme of utilizing two labels only, itis even possible to obtain the full sequence by employing only twolabelled bases per sequencing run. As was pointed out by Sauer et al.,“Detection and Identification of Single Dye Labelled MononucleotideMolecules Released From an Optical Fiber in a Microcapillary: FirstSteps Towards a New Single Molecule DNA Sequencing Technique,” Phys.Chem. Chem. Phys. 1:2471-77 (1999), which is hereby incorporated byreference, the sequence can be determined with 2 labels alone if onecarries out multiple sequencing reactions with the possible combinationsof the two labels. Therefore, in carrying out the process of the presentinvention, it is desirable to label long stretches of nucleic acid withat least 2 different labels.

Where sequencing is carried out by attaching the polymerase rather thanthe nucleic acid to the support, it is important that the enzymesynthesizes long stretches of nucleic acid, without the nucleicacid/protein complex falling apart. This is called processive nucleicacid synthesis. At least for the system using T7 DNA polymerase and dCTPcompletely replaced by coumarin-5-dCTP, the synthesis is fullyprocessive over at least 7300 basepairs (i.e., one polymerase moleculebinds to the ssDNA template and makes the entire second strand withoutfalling off even once). With one label, the process of the presentinvention can be carried out by watching the polymerase in real timewith base pair resolution and identifying the sequence profile of thatbase, but without knowing the other bases. Therefore, using fourdifferent labels would be most desirable for greater speed and accuracyas noted above. However, information from measuring incorporation ofnucleotides at a single molecule level, such as incorporation rates forindividual bases in a given sequence context, can provide a means offurther characterizing the sequence being synthesized. In respect toensuring processive synthesis for the second operational mode of thepresent invention, accessory proteins can be utilized to make thenucleic acid/protein complex even more processive than using the nucleicacid polymerizing enzyme alone. For example, under optimal conditions,T7 DNA polymerase is processive over at least 10,000 bases, whereas incomplex with the T7 helicase/primase protein, the processivity isincreased to over 100,000 bases. Kelman et al., “Processivity of DNAPolymerases: Two Mechanisms, One Goal” Structure 6: 121-125 (1998),which is hereby incorporated by reference. A single-stranded bindingprotein is also a suitable accessory protein. Processivity is especiallyimportant at concentrations of nucleotide analogs that are below thesaturation limit for a particular polymerase, because it is known thatprocessivity values for polymerases are decreased at limiting substrateconcentrations. See Patel et al., “Pre-Steady-State Kinetic Analysis ofProcessive DNA Replication Including Complete Characterization of anExonuclease-Deficient Mutant,” Biochemistry 30: 511-525 (1991), which ishereby incorporated by reference. Another possibility to ensureprocessivity is the development or discovery of a polymerase that isfully processive in the absence or at very low substrate concentrations(as is the case, e.g., for an elongating RNA polymerase/DNA complex). Incase the processivity is not sufficiently high, it is possible to attachboth the polymerase and the target nucleic acid molecule on the supportproximate to each other. This would facilitate the reformation of thecomplex and continuation of DNA synthesis, in case the sequencingcomplex falls apart occasionally. Non-processive polymerases can also beused in accordance with the present invention for the case where thetarget nucleic acid is bound to the support. Here, the same or adifferent polymerase molecule can reform the complex and continuesynthesis after dissociation of the complex.

One approach to carrying out the present invention is shown in FIG. 6.FIG. 6A shows a system for sequencing with reagent solution R positionedat surface 2 to which a primed target nucleic acid molecule complex isimmobilized. By confining illumination to a small area proximate to theactive site of polymerase extension, e.g. by focusing activatingradiation with the help of lens or optical fiber 6, nucleotide analogsthat become incorporated into the growing nucleic acid strand aredetected, because they are located within the region of illumination.FIG. 6B shows an enlarged section of the device, with the polymerizingcomplex in the region of illumination. The substrate concentration ischosen such that the number of nucleotide analogs in the surroundingarea in solution R are generally outside the illuminated region and arenot detected.

As shown in FIG. 6A, illumination source 10 (e.g., a laser) directsexcitation radiation by way of a dichroic beam splitter 8 through lens 6and surface 2 to the immobilized primed target nucleic acid complex.This excites the label immobilized to the complex with the resultingemitted radiation passing back through surface 2 and lens or opticalfiber 6. Dichroic beam splitter 8 allows passage of the emittedradiation to detector (or array of several detectors) 12 whichidentifies the type of emission. The detected emission information isthen directed to computer 14 where the nucleotide base corresponding tothe emission is identified and its identity stored. After multiplecycles of this procedure, the computer will be able to generate asoutput the sequence of the target nucleic acid molecule. Thecorresponding output of detection again corresponds to the scheme shownin FIG. 3, as explained above.

According to another embodiment of the present invention, illuminationand detection of fluorescence may be achieved by making the support forthe bound nucleic acid at the end of a first single-mode optical fibercarrying the excitation light. Either this and/or a second optical fibermay be used for collecting fluorescent photons. By transmitting theradiation of appropriate exciting wavelength through the firstsingle-mode optical fiber, the label will fluoresce and emit theappropriate fluorescent light frequency. The emitted fluorescent lightwill be partially transmitted into the second optical fiber andseparated spectrally such as by etched diffraction gratings on thefiber. The returned light spectrum identifies the particular boundnucleotide analog. Other techniques to deliver or collect light to thereaction site are conceivable, such as the use of waveguidedillumination or evanescent wave illumination, such as total internalreflection illumination. One or several illumination sources, deliveringone- or multiphoton excitation, can be employed. Suitable detectorsinclude avalanche photodiode modules, photomultiplier tubes, CCDcameras, CMOS chips, or arrays or combinations of several detectors.

Because there is likely to exist an upper limit to the concentration ofnucleotide analogs present in the observation volume that is correlatedto a permissible signal to background ratio and the ability todistinguish the particular nucleotide analog that is being incorporatedinto nucleic acid from the nucleotide analogs that are just diffusingaround the polymerase, it is possible that the sequencing procedure ofthe present invention must be carried out at concentrations below thesaturating limit for one or more nucleotide analogs.

For example, if conventional diffraction limited optics is used fordetection of fluorescence, the volume of observation is large so thatsubstrate concentrations in the range of nanomolar would have to be usedfor an acceptable background signal. This is far below the usual k_(m)of polymerases (usually in the range of μM), unless other means toreduce the background, such as lifetime discrimination as discussedabove (FIG. 5), or volume confinement techniques, as described below,are utilized to either “electronically” or physically reduce backgroundfluorescence contributions. In a conventionally focused laser beam, thefocal volume is approximately 0.2 μm³ (0.5 μm in diameter, 1.5 μm in theaxial direction), corresponding to about 0.2 fl. In order for only onefluorescent nucleotide analog to be present on average in the excitationvolume at any given time, the substrate concentration must be reduced toca. 10 nM, a concentration far below the k_(m) values of DNA polymerases(ca. 1-2 μM). See Polesky et al., “Identification of Residues Criticalfor the Polymerase-Activity of the Klenow Fragment of DNA-Polymerase-Ifrom Escherichia-coli,” J. Biol. Chem. 265:14579-14591 (1990) andMcClure et al., “The Steady State Kinetic Parameters andNon-Processivity of Escherichia coli Deoxyribonucleic Acid PolymeraseI,” J. Biol. Chem. 250:4073-4080 (1975), which are hereby incorporatedby reference. Thus, if the concentration of substrates is far below thek_(m), processivity of nucleic acid synthesis has to be ensured by oneof the above-mentioned possibilities. Alternatively, if the volume ofobservation can be reduced, a higher substrate concentration ispermissible, which naturally increases processivity values. Therefore,one objective of the present invention is concerned with an effectivereduction of the observation volume in order to reduce or preventbackground fluorescence caused by labelled free nucleotides and increaseprocessivity. This can be achieved in a number of ways.

One approach to reducing background noise involves electromagnetic fieldenhancement near objects with small radii of curvature.

Due to the so-called “antenna effect,” electromagnetic radiation isstrongly enhanced at the end of a sharp object, such as a metal tip.Using this procedure, the volume being enhanced roughly corresponds to asphere with a diameter that is close to the diameter of the tip. Thistechnique is disclosed in Sanchez, E. J., et al., “Near-FieldFluorescence Microscopy Based on Two-Photon Excitation with Metal Tips,”Phys. Rev. Lett. 82:4014-17 (1999), which is hereby incorporated byreference.

In carrying out the process of the present invention, a nucleic acidpolymerizing enzyme is positioned at the end of a metal tip with laserlight being directed on it, e.g. with a conventional objective lens.Because the effective illuminated volume can now be on the order of thesize of the polymerase itself, practically no fluorescence from thefluorescent nucleotides that are diffusing in the solution will bedetected. Furthermore, the residence time of diffusing molecules throughsuch a small volume is extremely short. However, incorporation of afluorescent nucleotide will be seen as a relatively long burst offluorescence, because that particular molecule will stay in this smallilluminated volume (until it is removed as explained above).

One approach to carrying out this embodiment of the present invention isshown in FIGS. 7A to B. FIG. 7A shows a system for sequencing withelectromagnetic field enhancement with reagent solution R positioned atsurface 2 to which a primed target nucleic acid molecule complex isimmobilized. As shown in FIG. 7B, a metal tip carrying a polymerase ispositioned in reagent solution R, creating a small region ofillumination around the immobilized polymerase upon illumination by lens6. By confining illumination to this small area, proximate to the activesite of polymerase extension, nucleotide analogs that becomeincorporated into the growing nucleic acid strand are detected, becausethey are positioned within the region of illumination. On the otherhand, nucleotide analogs in the surrounding area in solution R aregenerally outside this region and are not detected.

As shown in FIG. 7A, illumination source 10 (e.g., a laser) directs oneor multiphoton excitation radiation with a nonzero polarizationcomponent parallel to the tip by way of a dichroic beam splitter 8through lens 6 and surface 2 to the immobilized primed target nucleicacid complex. This excites the label immobilized to the complex with theresulting emitted radiation passing back through surface 2 and lens 6.Dichroic beam splitter 8 allows passage of the emitted radiation todetector 12 which identifies the type of emission. The detected emissioninformation is then directed to computer 14 where the nucleotide basecorresponding to the emission is identified and its identity stored.After multiple cycles of this procedure, the computer will be able togenerate as output the sequence of the target nucleic acid molecule. Thecorresponding output of detection again corresponds to the scheme shownin FIG. 3, as explained above. The principal difference to the casediscussed before is that the short peaks caused by randomly diffusingnucleotide analogs through the focal volume are now extremely short,because the volume of observation is so small. Therefore, this approachof reduction of observation volume also results in enhanced timeresolution in respect to incorporated nucleotides versus unincorporatedones. This is true for all of the other possibilities of volumeconfinement discussed further below.

In carrying out this procedure, the tips can be formed from a variety ofmaterials, e.g., metals such as platinum, silver, or gold. Thefabrication of the tip can be accomplished, e.g., by electrochemicaletching of wires or by ion-beam milling. See Sanchez, E. J., et al.,“Near-Field Fluorescence Microscopy Based on Two-Photon Excitation withMetal Tips,” Phys. Rev. Lett. 82:4014-17 (1999), which is herebyincorporated by reference.

The nucleic acid polymerizing enzyme can be attached to the end of thetip either by dipping the tip into a solution of nucleic acidpolymerizing enzyme molecules, applying an electric field at the tipwith charges attracting the nucleic acid polymerizing enzyme, or othertechniques of coupling (e.g., with linkers, antibodies etc.). Analternative mode of using electromagnetic field enhancement for thisscheme of sequencing is by positioning a bare tip in close proximity toan immobilized nucleic acid/nucleic acid polymerizing enzyme complex,rather than having the complex physically attached to the end of thetip. A population of complexes could, for example, be immobilized on aglass slide, and the tip is scanned over the surface until a usefulcomplex for sequencing is found. Suitable techniques for carrying outthis nanopositioning have been developed in the field of scanning probemicroscopy.

Another approach for reducing background noise while carrying out thesequencing method of the present invention involves the use ofnear-field illumination, as shown in FIGS. 8A-B. Here, as depicted inFIG. 8B, the primed target nucleic acid complex is immobilized onsurface 2 with opaque layer 16 being applied over surface 2. However,small holes 18 are etched into the opaque layer 16. When illuminatedfrom below, the light cannot penetrate fully through the holes intoreagent solution R, because the diameter of holes 18 is smaller than onehalf of the light's wavelength. However, there is some leakage whichcreates a small area of light right above surface 2 in hole 18, creatinga so-called near-field excitation volume. As shown in FIG. 8B, theprimed target nucleic acid complex is positioned in hole 18 where it isilluminated from below. By confining illumination to this smallnear-field area, incorporated nucleotide analogs, positioned within theregion of illumination, are detected. On the other hand, the quantity ofnucleotide analogs which do not serve to extend the primer are few innumber due to the small size of hole 18 and, to the small extentdetected, are easily distinguished from incorporated nucleotide analogsas described above.

The system for carrying out this embodiment is shown in FIG. 8A.Illumination source 10 (e.g., a laser) directs excitation radiation byway of dichroic beam splitter 8 through lens 6 and surface 2 to theimmobilized primed target nucleic acid complex. This excites the labelimmobilized to the complex with the resulting emitted radiation passingback through surface 2 and lens 6. Dichroic beam splitter 8 allowspassage of the emitted radiation to detector 12 which identifies thetype of emission. The detected emission information is then directed tocomputer 14 where the nucleotide base corresponding to the emission isidentified and its identity stored. After multiple cycles of thisprocedure, the computer will be able to generate as output the sequenceof the target nucleic acid molecule.

As a suitable alternative using near-field excitation volumes, thenear-field volume can also be generated by the use of one or manytapered optical fibers commonly used in scanning near-field microscopy.

Nanofabrication is another technique useful in limiting the reactionvolume to reduce the level of background fluorescence. This involvesconfinement of the excitation volume to a region within a nanochannel.Here, confinement is possible in two of three spatial dimensions. Areaction vessel with a volume much smaller than focal volumes attainablewith far-field focusing optics is fabricated on a silicon or fusedsilica wafer from optically transparent materials. Turner et al.,“Solid-State Artificial Gel for DNA Electrophoresis with an IntegratedTop Layer,” Proceedings of SPIE: Micro- and Nano-Fabricated Structuresand Devices for Biomedical Environmental Applications 3258:114-121(1998), which is hereby incorporated by reference. The technique takesadvantage of a polysilicon sacrificial layer to define the workingcavity of the channels. Stern et al., “Nanochannel Fabrication forChemical Sensors,” J. Vac. Sci. Technol. B15:2887-2891 (1997) and Chu etal., “Silicon Nanofilter with Absolute Pore Size and High MechanicalStrength,” Proc. SPIE—Int. Soc. Opt. Eng. (USA) 2593: 9-20 (1995), whichare hereby incorporated by reference. The floor, ceiling, and walls ofthe channels are made of silicon nitride, which is deposited conformallyover a patterned polysilicon sacrificial layer. The sacrificial layer isthen removed with a high-selectivity wet chemical etch, leaving behindonly the silicon nitride. This technique has demonstrated precisecritical dimension (CD) control over a wide range of structure sizes.The height of the polysilicon layer can be controlled to within 5 nmover an entire device, and the lateral dimensions are limited in sizeand CD control only by the lithography technique applied. Thenanostructure can have a punctuate, acicular, or resonant configurationto enhance label detection.

FIGS. 9A-B show a nanofabricated system in accordance with the presentinvention. Shown in FIG. 9B is an enlarged view of the cross-section ofthe nanochannel, with reagents R located only in confined area 102,which is created by the channel walls 104 and 106. The primed targetnucleic acid molecule complex is positioned within confined area 102. Asa result, when excitation light passes through confined area 102, thelabel of the incorporated nucleotide analog is excited and emitsradiation which is detected and identified as corresponding to aparticular nucleotide base added to the sequence of the extendingprimer. By passing the reagents through confined area 102, the quantityof nucleotide analogs which do not extend the primer are few in numberat any particular point in time. To the small extent such mobileentities are detected, they are easily distinguished from immobilizedmoieties as described above.

FIG. 9A shows a system for carrying out the nanochannel embodiment ofthe present invention. Illumination source 10 (e.g., a laser) directsexcitation radiation by way of dichroic beam splitter 8 through lens 6and nanochannel 106 to the immobilized primed target nucleic acidcomplex. This excites the label immobilized to the complex with theresulting emitted radiation passing back through lens 6. Dichroic beamsplitter 8 allows passage of the emitted radiation to detector 12 whichidentifies the type of emission. The detected emission information isthen directed to computer 14 where the nucleotide base corresponding tothe emission is identified and its identity stored. After multiplecycles of this procedure, the computer will be able to generate asoutput the sequence of the target nucleic acid molecule.

FIGS. 10A-B show systems for supplying reagents to a nanofabricatedconfinement system in accordance with the present invention. In FIG.10A, the reagents, which include dATP, dCTP, dGTP, dUTP, the nucleicacid source, and buffer are held in separate reservoirs and connectedthrough separate conduits to manifold 200 where the reagents are mixedtogether before entering nanochannel 202. The components of this systemupstream and downstream of nanochannel 202 can be combined as amicrostructure. In the process of passing rapidly through nanochannel202, the reagents move rapidly through reaction zone 204 where thesequencing procedure of the present invention is carried out. Fromnanochannel 202, the residual reagents R pass through outlet 206. Thesystem of FIG. 10B is generally similar to that of FIG. 10A, but theformer system is on a single chip with pads to connect the system tofluid reservoirs. In particular, the reservoir for each of the reagentsis coupled to the chip 208 via inlet pads 210 a-f, while the outlet fordischarged reagents is connected to pad 212.

Nanofabricated channels of 75 nm width and 60 nm height have beenmanufactured with excellent optical transparency and used for DNA flowcontrol. See Turner et al., “Solid-State Artificial Gel for DNAElectrophoresis with an Integrated Top Layer,” Proceedings of SPIE:Micro- and Nano-Fabricated Structures and Devices for BiomedicalEnvironmental Applications 3258:114-121 (1998), which is herebyincorporated by reference. By placing the nucleic acid synthesis complexinto a channel of depth z=25 nm, minimizing the x-dimension of thefocused laser beam to ca. 300 nm, and fixing the y-dimension by thechannel width at 100 nm, the effective volume of observation can bereduced to 7.5×10⁻⁴ μm³, corresponding to 0.75 attoliters. Here, theconcentration for only one substrate molecule to be present in theexcitation volume amounts to 2 μM, a substrate concentration well withinthe range of rapid and efficient nucleic acid polymerization. Moreover,since there are four different nucleotide analogs, each to bedistinguished, the effective substrate concentration for the polymeraseis four times higher. If a smaller effective volume of observation isrequired, the y-dimension in the flow direction can be reduced to about100 nm by illumination with the interference pattern of two objectivesat about 90° axial angles as in theta microscopy. See Stelzer et al., “ANew Tool for the Observation of Embryos and Other Large Specimens:Confocal Theta Fluorescence Microscopy,” J. Microscopy 179:1-10 (1995),which is hereby incorporated by reference.

To excite the labels, activating energy is focused proximate to theactive site of polymerase extension (i.e. where the polymerase islocated). To the extent this active site moves during extension (e.g.,as a result of movement by the polymerase), the focus of the activatingenergy is also moved.

A necessary consideration is the choice between one-photon andmultiphoton excitation of fluorescence. Multiphoton excitation providessome powerful advantages, but it is more complex and more expensive toimplement. Multiphoton excitation fluorescence utilizing simultaneousabsorption of two or more photons from bright, femtosecond infraredpulses generated by ultrafast solid state mode locked lasers providesthe most promising approach. See Denk et al., “2-Photon Laser ScanningFluorescence Microscopy,” Science 248:73-76 (1990), which is herebyincorporated by reference. Sensitivity to single molecule fluorescenceis routinely obtained and is temporally resolvable to the microsecondlevel with fluorescence lifetimes measurable with reasonable accuracyfor single molecules. See Mertz et al., “Single-Molecule Detection byTwo-Photon-Excited Fluorescence,” Optics Lett. 20:2532-2534 (1995) andEggeling et al., “Monitoring Conformational Dynamics of a SingleMolecule by Selective Fluorescence Spectroscopy,” Proc. Natl. Acad. Sci.USA 95:1556-1561 (1998), which are hereby incorporated by reference.

The ideal fluorescent signal for single molecule sequencing consists oftime resolved bursts of distinguishable fluorescence as each nucleotideis bound. Thus, in the ideal situation, a time-resolved train of colorresolved fluorescent bursts could be obtained if nucleotides were boundat distinguishable intervals as described in FIG. 3. Full resolution ofthe time sequence of events therefore offers the best backgroundreduction and reliable possibility for nucleotide recognition. Sincewith the currently available polymerases, labelled nucleotides are mostlikely added no faster than at 1 millisecond intervals, it should bepossible that all of the detected fluorescence photons from eachlabelled nucleotide can be accumulated and removed before the nextfluorescent nucleotide is bound. This ideal burst-gap-burst sequence isrealized although actually every molecular kinetic step ofpolymerization involves the stochastic Poisson process. For a singlePoisson process, the most probable time delay between events is zeroalthough the average delay would be larger than zero. However, theprocess of incorporation of a single dNTP into DNA by DNA polymerase isa sequential multistep process of at least 5 different events. See Patelet al., “Pre-Steady-State Kinetic Analysis of Processive DNA ReplicationIncluding Complete Characterization of an Exonuclease-Deficient Mutant,”Biochemistry 30: 511-525 (1991). The sequential summation of these stepswill result in a most likely time delay larger than zero. Therefore, thephoton bursts are not very likely to overlap.

For conventional fluorophores, about 10⁵ photons per fluorophore will beemitted before photobleaching. Detection of (at most) 1% of the emissionyields about 10³ photons for a relative noise uncertainty of 3%.Background due to free nucleotides is reduced to a nearly negligiblelevel by the schemes discussed above, e.g., by limiting the size of thefocal volume to contain only about one free labelled nucleotide, withvery short dwell times.

The expected detection level is about 10³ photons from each labellednucleotide, in about 10⁻³ s. This is an acceptable counting rate, ˜10⁶Hz, and an acceptable fluorophore excitation rate at about one tenth ofsinglet excited state saturation. This fluorescence excitation creates adetected burst of ˜10³ photons in about 1 ms at the characteristicwavelength for each labelled nucleotide, leaving, on average, a gap ofabout 1 ms before the next nucleotide is added, well within the averagetime intervals between nucleotide addition at probably more than onemillisecond. Possible burst overlaps can be analyzed and resolved by theanalytical treatment of continuous measurements of data in time coherentsequences in (at best) 4 channels for most accurate sequencing results.With the photon statistics available in the experimental design andrecently developed coupled multichannel analyzers and operationalsoftware, error rates can be made acceptable with 4 labelled nucleotidesor with the strategies involving a smaller number of labels as outlinedabove.

Spectral resolution of four fluorophores identifying the nucleotides canbe achieved with two-photon excitation by infrared pulses. All 4fluorophores can be simultaneously excited due to the wide excitationbands usually characteristic of two-photon excitation. See Xu et al.,“Multiphoton Excitation Cross-Sections of Molecular Fluorophores,”Bioimaging 4:198-207 (1996), which is hereby incorporated by reference.Alternatively, multiple excitation sources can be used in combination orby fast switching to illuminate the sequencing complex if necessary.Spectral separation is accomplished with conventional interferencefilters but emission spectra may overlap, complicating the timecorrelation analysis and perhaps requiring cross correlation of the 4color channels for correction. If compatibility of fluorophores with thenucleic acid polymerizing enzyme limits the applicability of suitabledye sets, a combination of techniques can be applied to distinguish thelabels.

Another potential way to distinguish incorporation of a nucleotide intothe growing nucleic acid strand consists of measuring changes influorescence lifetime. Fluorescence lifetime of an oligonucleotidepyrene probe has been observed to vary in a sequence-dependent mannerupon DNA attachment. See Dapprich J, “Fluoreszenzdetection MolekularerEvolution (Fluorescence Detection of Molecular Evolution),”Dissertation, Georg-August-Univ., Goettingen, Germany (1994), which ishereby incorporated by reference. Photophysical interactions between thefluorophore and the base result in characteristic fluorescence decaytimes, and can also be used to differentiate the bases, as discussedabove. Lifetime determination and discrimination on the single moleculelevel has recently been demonstrated so that discrimination betweenbases being incorporated and freely diffusing nucleotides could becarried out by fluorescence lifetime measurements. See Eggeling et al.,“Monitoring Conformational Dynamics of a Single Molecule by SelectiveFluorescence Spectroscopy,” Proc. Natl. Acad. Sci. USA 95:1556-1561(1998), which is hereby incorporated by reference.

Time correlated measurements in four fluorescence wavelength channelscan be used effectively in carrying out the process of the presentinvention. Overlap of emission spectra may allow signals from onefluorophore to enter several channels but the relative count rate andtiming identifies the label correctly. Simultaneous signals from anincorporated labelled nucleotide and a free label are distinguishable bythe time duration and magnitude of the bursts, which are limited for thefree label. Label ambiguity can be further reduced by utilization offluorescence decay time measurements which can be realized with theavailable 0.1 ns resolution of time delays for fluorescence photonemission after each femtosecond laser excitation pulse. The fluorescencephoton emission and photobleaching processes themselves are alsostochastic processes but involve sufficiently disparate quantumefficiencies that error rates should be negligible.

In rejecting background from the freely diffusing or flowing labellednucleotides, the very short dwell time of any individual free nucleotidein the focal volume is advantageously used. The characteristic diffusiontime for a free nucleotide analog across the open dimension of the focalvolume (in the worst case of non-interferometric far-field illumination)will be τ_(D)˜y²/4D˜2×10⁻⁵ sec, with y being the focal volume dimensionand D the diffusion coefficient. An iontophoretic flow velocity of 1cm/s is sufficient to keep its short bursts of fluorescence to less than10⁻⁵ sec and reduce the photon numbers by an order of magnitude. Thiswill assure discrimination against free nucleotides and identify thetime series of bursts representing the nucleic acid sequence, providedthe nucleotide analog concentrations are appropriately low as discussed.Magde et al., “Thermodynamic Fluctuations in a ReactingSystem—Measurement by Fluorescence Correlation Spectroscopy,” Phys. Rev.Lett. 29:705-708 (1972) and Maiti et al., “Measuring SerotoninDistribution in Live Cells with Three-Photon Excitation,” Science275:530-532 (1997), which are hereby incorporated by reference.Discrimination can be improved by utilizing volume confinementtechniques or time-gated detection, as discussed above.

Detection of fluorescence resonance energy transfer (FRET) from a donorfluorophore (e.g., a donor attached to the polymerase) to adjacentnucleotide analog acceptors that are incorporated into the growingnucleic acid strand suggests a further elegant possibility of loweringbackground from incorporated nucleotides. FRET only reaches very shortdistances including about 20 nucleotides and decays at the reciprocalsixth power of distance. The excited donor molecule transfers its energyonly to nearby acceptor fluorophores, which emit the spectrally resolvedacceptor fluorescence of each labelled nucleotide as it is added.Already incorporated nucleotides farther away from the donor would notcontribute to the fluorescent signal since distance and orientationconstraints of energy transfer reduce the effective range of observationto less than 60 Å, thereby effectively eliminating backgroundfluorescence from unincorporated nucleotides. Without photobleaching,the method requires high sensitivity since repeat nucleotides leave therange of FRET at the same rate that new nucleotides are added, possiblycreating sequence recognition ambiguity. Photobleaching or photochemicalcleavage, or their combination as discussed above could resolve theproblem. Photobleaching of the donor molecules using FRET can be avoidedif it is the template nucleic acid that is attached and the donorbearing nucleic acid polymerizing enzyme is periodically replaced.

A final important consideration for the success of the present inventionconcerns the stability of the protein/nucleic acid complex in activatingradiation, such as tightly focussed laser beams. It is not expected thatthe enzyme is affected by the excitation illumination, becausewavelengths are chosen at which proteins do not absorb, the stability ofthe polymerase in the laser beam should be sufficiently high to allowfor accurate sequencing runs over long read lengths. Previousinvestigations exposing enzymes to strong laser light have examinedphotodamage and loss of function. Immobilized RNA polymerase/DNAcomplexes showed inactivation times of 82±58 s for 1047 nm Nd:Y laserlight of 82 to 99 mW laser power focused at the protein, correspondingto intensities of approximately 10⁸ W/cm². Other studies on theactomyosin or kinesin systems indicated similar stability. Both DNA andbiotin-avidin linkages have been shown to be photostable in opticaltraps. See Yin et al., “Transcription Against an Applied Force,” Science270: 1653-1657 (1995), Svoboda et al. “Direct Observation of KinesinStepping by Optical Trapping Interferometry,” Nature 365: 721-727(1993), and Molloy et al., “Movement and Force Produced by a SingleMyosin Head” Nature 378: 209-212 (1995), which are hereby incorporatedby reference. For fluorescence detection of nucleotide analogs accordingto the present invention, laser powers (intensities) typical of FCSmeasurements are expected, on the order of 0.1 mW (10⁵ W/cm²) forone-photon and 1 mW (10⁶-10⁷ W/cm²) for two-photon excitation, therebybeing significantly lower than in the case of optical tweezers describedabove. Enzyme stability should therefore be higher, moreover, with therapid speed of sequencing proposed by this method (e.g., 100 bp/s), even80 s are sufficient to determine the sequence of 8 kb nucleic acid.

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method of sequencing a target nucleic acid molecule, comprising:(a) providing a complex comprising a polymerase enzyme, a target nucleicacid molecule, and a primer, wherein the complex is immobilized on asupport, whereby the complex is within an observation volume of adetector; (b) exposing said complex to a plurality of types of labelednucleotides or labeled nucleotide analogs for incorporation into agrowing nucleic acid strand complementary to the target nucleic acid,each type of labeled nucleotide or labeled nucleotide analog comprisinga fluorescent label that is distinguishable from the other types of saidplurality, wherein the fluorescent label is attached to a terminalphosphate group of the nucleotide or nucleotide analog; and (c)detecting with the detector the label on the nucleotide or nucleotideanalog while the labeled nucleotide or nucleotide analog is retained inthe complex for a period of time sufficient for incorporating into thegrowing strand, thereby identifying the nucleotide or nucleotide analogincorporated by the polymerase enzyme into the growing strand, wherebythe time duration of the signal from labeled nucleotides or nucleotideanalogs that become incorporated is distinguished from freely diffusinglabels by a longer retention in the observation volume for thenucleotides or nucleotide analogs that become incorporated than for thefreely diffusing labels.
 2. The method of claim 1, wherein saiddetecting the label on the nucleotide or nucleotide analog is effectedby providing an excitation radiation to said label.
 3. The method ofclaim 1, wherein said label comprises a fluorescent nanoparticle.
 4. Themethod of claim 1, wherein the label is covalently attached to thenucleotide or nucleotide analog.
 5. The method of claim 1, wherein thepolymerase is a DNA polymerase.
 6. The method of claim 1, wherein thecomplex is formed by immobilizing the target nucleic acid or the primeronto the support, followed by exposing the immobilized target nucleicacid and/or primer to a polymerase.
 7. The method of claim 1, whereinthe complex is formed by immobilizing the polymerase enzyme onto thesupport, followed by exposing the immobilized polymerase enzyme to saidtarget nucleic acid and/or said primer.
 8. A method of sequencing aplurality of target nucleic acid molecules in parallel, comprising: (a)providing a field of immobilized complexes, each of which comprises apolymerase enzyme, a target nucleic acid molecule, and a primer, whereinan individual complex in said field is within an observation volume of adetector and is optically resolvable by the detector; (b) exposing saidfield of immobilized complexes to a plurality of types of labelednucleotides or labeled nucleotide analogs for incorporation into growingnucleic acid strands complementary to the target nucleic acids, eachtype comprising a fluorescent label that is distinguishable from theother types of said plurality, wherein the fluorescent label is attachedto a terminal phosphate group of the nucleotide or nucleotide analog;(c) directing excitation radiation to the field; (d) detecting with thedetector the label on the nucleotide or nucleotide analog while thelabeled nucleotide or nucleotide analog is retained in an individualcomplex for a period of time sufficient for incorporating into thegrowing strand, thereby identifying the nucleotide or nucleotide analogincorporated by the polymerase enzyme into the growing strand, wherebythe time duration of the signal from labeled nucleotides or nucleotideanalogs that become incorporated is distinguished from freely diffusinglabels by a longer retention in the observation volume for thenucleotides or nucleotide analogs that become incorporated than for thefreely diffusing labels; (e) recording the identity and position of theincorporated nucleotide or nucleotide analog based upon said opticalsignal, thereby sequencing the plurality of target nucleic acidmolecules in parallel.
 9. The method of claim 8, wherein said detectingthe label on the nucleotide or nucleotide analog is effected byproviding an excitation radiation to said label.
 10. The method of claim8, wherein said label comprises a fluorescent nanoparticle.
 11. Themethod of claim 8, wherein the label is covalently attached to thenucleotide or nucleotide analog.
 12. The method of claim 8, wherein thepolymerase is a DNA polymerase.
 13. The method of claim 8, wherein theindividual complex is formed by immobilizing the target nucleic acid orthe primer onto the support, followed by exposing the immobilized targetnucleic acid and/or primer to a polymerase.
 14. The method of claim 8,wherein the individual complex is formed by immobilizing the polymeraseenzyme onto the support, followed by exposing the immobilized polymeraseenzyme to said target nucleic acid and/or said primer.